TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4

Abstract

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liverspecific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

“…For example, Mediator is a component of the scaffold, which remains once the Pol II from the pioneer PIC has progressed into the elongation mode (24), and likely plays a role in facilitating subsequent cycles of reinitiation. Potentially related, there also is strong evidence for a postrecruitment function for the Mediator (14,15). Further complicating straightforward conclusions regarding Mediator mechanisms are newer observations, which have been described here and also previously published (23), that excess TFIIB allows the Mediator requirement to be bypassed.…”

“…Although Mediator can further stimulate this activity, the basis of the high Mediatorindependent activity has remained unclear. On the other hand, upon immunodepletion of Mediator from unfractionated nuclear extract (which presumably more closely approximates the distribution of cellular factors), both activator-dependent and basal transcription are abolished and can be effectively restored upon provision of pure Mediator (10,12,14). These observations suggest that in the context of the extract, and thus the cell, activity of any PICs assembled in the absence of Mediator is subject to some restriction.…”

“…Purified Mediator [either intact TRAP/SMCC (20,21) or PC2, the form that mainly lacks the TRAP240/MED13-TRAP230/MED12-CDK8-CycC subcomplex (13,22)] could be shown to potentiate both basal and activated transcription. Similarly, in a Mediator-depleted nuclear extract system, Mediator dependence of basal and activated transcription was readily demonstrable (10,12,14,17). Thus, whereas in the purified system the Mediator boosts transcription levels that are already quite high, in the cruder system the Mediator dependence is essentially absolute.…”

“…Importantly, in contrast to chromatin coactivators, Mediator can facilitate DNA-templated transcription, and thus acts relatively late in the overall activation pathway on natural templates (8). Several lines of evidence suggest that Mediator function is manifested both at the level of the initial establishment of the PIC as well as its postrecruitment function (5,7,(9)(10)(11)(12)(13)(14)(15).…”

The Mediator Complex in Plants: Structure, Phylogeny, and Expression Profiling of Representative Genes in a Dicot (Arabidopsis) and a Monocot (Rice) during Reproduction and Abiotic Stress

“…Despite a rigorous search, only the red alga C. merolae had a detectable Med1 subunit in the plant kingdom. Med1 is the target for the aryl-hydrocarbon receptor (Wang et al, 2004) and several liganded nuclear receptors via its LxxLL (Leu-X-X-Leu-Leu) motif (Yuan et al, 1998;Malik et al, 2002). CmMed1_1 has two LxxLL motifs at positions 219 and 384.…”